In the past years immunological detection methods have become very important in diagnostics. These methods enable analytes to be detected in biological samples. These analytes for example include drugs, hormones, proteins, infectious agents, microorganisms and antibodies to these analytes. These pathogens are either detected directly or indirectly in particular to detect infections by microorganisms such as bacteria, fungi or viruses. This means that, depending on the infection, the pathogen is diagnosed by an antigen test or the antibodies which have been formed specifically as an immune response to the pathogen are detected.
In all immunological detection reactions a specific binding reaction occurs between the substance which it is intended to detect (analyte) and at least one specific binding partner which specifically reacts with the analyte or specifically binds it. In this process the analyte and the specific binding partner form a specific binding pair which is in general a complex between an antigen and an antibody or an antibody fragment. In this process more than one analyte or more than one binding partner can react together in each reaction. These specific binding reactions can be detected in various ways. In general one binding partner of the specific binding reaction is labelled. Common labels are chromogens, fluorophores, substances capable of chemiluminescence or electrochemiluminescence, radioisotopes, haptens, enzyme labels or substances which in turn can form a specific binding pair such as biotin/streptavidin.
A serious problem in immunoassays is that undesired interactions and nonspecific binding reactions can take place between the specific binding partners of the immunoassay and the sample components. Such interactions usually lead to an increase of the background signal, a stronger scattering of the signals and thus a reduced sensitivity and specificity of the respective test.
Depending on the type of interference caused by nonspecific interactions, false-positive or false-negative test results can also occur.
False-negative results can occur when a substance is present in the sample which masks the analyte to be detected so that the specific detection reagents, for example an antibody, can no longer bind to the analyte.
False-positive test results are a particularly major problem. This means that a positive signal is obtained in the test although the analyte is absent. Thus, especially when diagnosing infectious diseases, the situation should not occur that samples of healthy, non-infected patients give a false-positive result in the test. In the diagnosis of HIV infections the requirements made by the approval authorities for the clinical specificity of diagnostic tests for the detection of anti-HIV antibodies is larger than 99.5%. This means that in a group of normal donors (samples from non-infected persons) no more than 5 false-positive samples may occur in 1000 samples. The false-positive reactions which nevertheless occur are caused by nonspecific substances which, depending on the test method, bind to the antigens, e.g. HIV antigens that are used for the antibody detection and then, like antibodies to the infectious agent to be detected, are falsely detected as positive by the detection system. These nonspecifically reacting substances are often antibodies. Various attempts have already been described in the prior art to reduce these nonspecific interactions in immunoassays which lead to false test results. Thus it has been known for a long time that various carbohydrate components and proteins, protein mixtures, certain protein fractions and hydrolysates thereof can reduce nonspecific interactions between the test components and the analyte in immunoassays (see for example Robertson et al., J. Immunol. Meth. 26, 1985; EP-A-0 260 903; U.S. Pat. No. 4,931,385). A disadvantage of using crude protein fractions and crude hydrolysates is that components contained therein can in turn cause other interferences of the test. Furthermore hydrolysates that are produced enzymatically can be contaminated with the proteases used for their manufacture and usually do not have a uniform quality since the enzymatic cleavage is difficult to control. Protease contaminants can attack test components and even in small amounts can lead to an impairment of the test functions and storage stability.
The use of chemically modified proteins and especially of succinylated or acetylated proteins has also been described for reducing nonspecific interactions in immunoassays (U.S. Pat. No. 5,051,356; EP-A-0 525 916). However, many of the false-positive results in antibody tests from serum samples cannot be avoided using these substances.
EP-A-0 331 068 and WO 91/06559 describe the use of polymerized immunoglobulins, in particular IgG, to reduce specific interfering factors such as e.g. rheumatoid factors. However, they do not enable the satisfactory elimination of all interfering interactions. Moreover, the addition of nonspecific human immunoglobulins in tests for human antibodies can lead to an increase of the blank value. Furthermore the isolation of human or animal IgG is complicated and expensive.
Avidin and streptavidin as well as derivatives thereof are described in WO 95/23801 as interference-eliminating agents which mainly reduce nonspecific interactions of the sample components with a streptavidin or avidin solid phase in heterogeneous immunoassays. These interference-eliminating agents cannot eliminate interference by substances that do not interact with the solid phase but rather nonspecifically bind to the usual immunological test components.